Previously, we have isolated and chemically characterized a FMRF- like peptide of bovine brain. This peptide, phe-leu-phe-gln-arg- phe-NH2), can decrease analgesic effect of morphine and is highly localized in spinal cord and periaqueductal gray areas. In this study, the locations of F-8-F-NH2 in periaqueductal gray areas of rat and bovine brains were investigated immunohistochemically using polyclonal and monoclonal antibodies. The polyclonal antiserum cross-reacts slightly with NPY (is less than 0.1%), while the monoclonal antibody is devoid of such cross-reactivity. F-8-F-NH2 immunoreactivity is localized in nerve fibers running vertically throughout periaqueductal gray area. In horizontal sections F-8- F-NH2 immunoreactive nerves form dense networks in posterolateral periaqueductal gray areas. F-8-F-NH2 immunoreactive cell bodies were not detected in the periaqueductal gray areas even in the rats which were treated with colchicine to block axonal transport. The immunostaining was abolished by preabsorption with F-8-F-NH2 but not by NPY which is structurally similar to NPY in its c-terminal dipeptide. The results of this study indicate that there are specific F-8-F-NH2 containing verve fibers in periaqueductal gray areas and they seem to originate from other parts of the brain.